The biosynthesis of lysosomal enzymes in cultured human fibroblasts has been studied by labeling the cultures with radioactive leucine, mannose or phosphate, immunoprecipitating the radioactive enzymes and examining them by flourography after electrophoresis in sodium dodecyl sulfate. Three enzymes, beta-hexosaminidase (A and B isozymes), alpha-glucosidase and cathepsin D were found to be synthesized as precursors of larger molecular weight and subsequently trimmed to the size of tissue enzymes. In normal cells, the three lysosomal enzymes were found to contain phosphate as mannose 6-phosphate residues; phosphorylation was deficient in enzymes produced by fibroblasts of patients with I-cell disease. The mannose 6-phosphate constitutes a portion of the recognition marker for receptor binding and pinocytosis. By a novel screening technique, mutants of lysosomal enzymes have been isolated in CHO cells and used to demonstrate an uptake system recognizing mannose 6-phosphate, similar to that of human fibroblasts.